Pergamon Int. J. Hydrogen Energy, Vol. 19, No. 5, pp. 437-440, 1994

Copyright © 1994 International Asso~ation for Hydrogen Energy Elsevier Science Ltd

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HYDROGEN PRODUCTION BY RHODOPSEUDOMONAS AT THE

EXPENSE OF VEGETABLE STARCH, SUGARCANE JUICE AND WHEY

S. P. SINGH,* S. C. SRIVASTAVA and K. D. PANDEY

Centre of Advanced Study in Botany, Banaras Hindu University, Varanasi-221 005, India

(Received for publication 7 June 1993)

Abstract--Four local strains of Rhodopseudomonas sp. (BHU strains 1-4) evolved hydrogen at the expense of potato starch, sugarcane juice and whey (1% in each case) in the presence of light (2 klux), under anaerobic conditions (argon/CO2, 95/5, v/v). Among the three substrates, sugarcane juice supported the maximum level of H2 production, followed by potato starch and whey at the rates of 45, 30 and 25 #1 H2 h- 1 rag- 1 bacterial cell dry weight, respectively. Although elevated temperature (45°C) suppressed H 2 evolution by strains 1, 2 and 3, the thermotolerant strain (BHU strain 4) has shown encouraging results. Alginate-immobilized cells under an identical experimental regime, exhibited an almost one-and-a-half times improvement in H2 production in the cases of all the above substrates over their free cell counterpart. Preliminary experiments have shown the presence of amylase in all the bacterial strains and work is in progress to characterize this enzyme so that the system could be used for efficient consumption of starch-based agro- products.

INTRODUCTION

Hydrogen (clean energy) has been recognized by now to be the only environmentally safe energy source and alternative to fossil fuel. Among the various physical and biological routes known for H 2 production, photosyn- thetic bacteria represent a futuristic approach with an appreciable extent of light-conversion efficiency I'1, 2]. The nonsulphur photosynthetic bacteria, in particular, evolve hydrogen at the expense of simple organic acids or sugars, and their performance depends on factors such as availability of substrates, light irradiance and tempera- ture. To minimize the cost of H2 production, various raw materials of agro-based industry or others like whey, sugar refinery waste, sugarcane juice, corn pulp or mo- lasses have been utilized for growth and hydrogen pro- duction by Rhodopseudomonas [3, 4]. The bacterial consumption of raw starch depends on the presence of amylase [5], as the two strains (T-14 and T-20) of R. gelatinosa when supplied with either soluble starch or raw starch, evolved H 2 with an appreciable rate, especially with raw corn and potato starch [6-1. Whey, a by-product of the milk processing industry, has also been used as the efficient substrate for H2 production by photosynthetic bacteria [7, 8]. In previous communica- tions, we described sustained H 2 production by immobi- lized cells by a local strain of Rhodopseudomonas at the expense of potato starch [9] and also the isolation of high-temperature strains showing significant H 2 produc- tion even up to 45°C [10].

*Corresponding author.

In the present paper, we compare H 2 production by normal and temperature tolerant strains of nonsulphur photosynthetic bacterium Rhodopseudomonas sp. at the expense of potato starch, sugarcane juice and whey using free and immobilized cells.

EXPERIMENTAL

lnocula

The bacterial strains were isolated from the root zones of AzoUa, Salvinia, Eichhornia and those associated with deep-water rice plant roots [10]. These strains have been tentatively designated as Rhodopseudomonas sp. BHU strains 1-4, respectively (BHU for Banaras Hindu Uni- versity, India).

Growth medium

The basal medium as prescribed by Pfennig [11] was used with slight modifications C9]. All chemicals were of laboratory reagent grade obtained from BDH (U.K. or India). The pH of the growth medium was adjusted to 6.8. The concentrated bacterial suspensions were trans- ferred anaerobically to fresh growth medium in rubber- stoppered glass bottles having a gas phase of 95% argon and 5% CO 2. Cultures were grown under tungsten illumination (2 klux) at 33°C with periodic shaking.

Substrates Sugarcane from local agricultural fields was crushed

and squeezed to procure juice, followed by filtration

437

438 S.P. SINGH et al.

through cheese cloth to remove the suspended particles. Potato extract was prepared as described by Singh et al. [9]. Whey was prepared by precipitation of milk protein with citric acid followed by filtration of coagulated proteins through muslin cloth. The initial and subse- quent pH value for all the substrates used was adjusted to 6.8.

Cell immobilization

The exponential phase bacterial cells were concen- trated through centrifugation and immobilized in cal- cium alginate as described by Singh et al. [9]. The resulting beads were washed twice with sterile double- distilled water before transfer to fresh growth medium.

Test for amylase activity

The amylase activity in sonicated cell extract was determined by the method of Bernfeld [121 based on the determination of reducing sugars liberated in the reac- tion mixture.

Total oroanic carbon

Total organic carbon in the different substrates was measured by Walkley and Black's rapid titration method [13].

Measurement o f hydrooen production

Hydrogen production was determined by gas chroma- tography (Tracor 540, U.S.A., TCD mode) as described before [9].

RESULTS AND DISCUSSION

The four different local bacterial strains, namely BHU strains 1-4 of Rhodopseudomonas sp. isolated from root zones of Azola, Salvinia, Eichhornia and deep-water rice plant, respectively [10], showed photoevolution of hy- drogen to the extent of 12, 10, 9 and 12 gl H 2 h - i mg- dry weight, respectively (33°C). All these strains have been compared for their H2-production efficiency at the expense of potato starch, sugarcane juice and whey containing the percentage of total organic carbon as 3.5, 5.3 and 3.0, respectively, and the observations presented in Table 1. Whereas potato starch showed maximum two-and-a-half times stimulation of H 2 production by free cells over control sets within 48 h of incubation (light-anaerobic), sugarcane juice-based H2 production recorded a maximum of a four-fold enhancement; whey, under similar experimental conditions, was a poor sub- strate, as evident from a nearly two-fold increase in H 2 production rate. Such a comparison also reflected strain- specific differences as BHU strains I and 4 proved superior to strains 2 and 3. There are reports that when

Table 1. Comparison of hydrogen production by frc¢ and immobilized cells of Rhodopseudo- monas sp. (BHU strains 1-4) at the expense of potato starch, sugarcane juice and whey 0%

in each case, 33 ° and 45°C) at 48 h incubation

H2 production ~1 Hz h- t rag-1 dry weight)

33°C 45°C

Strain no. Free cells Immobilized cells Free cells Immobilized cells

BHU strain 1 Control 12 18 7 10 Potato starch 30 43 18 26 Sugarcane juice 45 68 27 39.5 Whey 25 38 14 21

BHU strain 2 Control 10 14 4 6.5 Potato starch 19 27 8.5 13 Sugarcane juice 32 47.5 15.5 23 Whey 14 20 6.5 10

BHU strain 3 Control 9 ! 3.5 2.5 4 Potato starch 14.5 20.5 4.5 6.5 Sugarcane juice 26 38 8.5 12.5 Whey 13.5 20 3.5 5

BHU strain 4 Control 12 20 15 24 Potato starch 28 42 38 56.6 Sugarcane juice 47 72 58 88 Whey 27 41 34 58

HYDROGEN PRODUCTION BY RHODOPSEUDOMONAS 439

sugar-rich preparations from sugarcane, corn or cellulose sources were added to methanogenic cultures, CO2 and H2 were produced in place of methane at neutral pH [14]. Currently, we are looking into possible reasons for the maximum H 2 production in sugarcane juice-supple- mented cultures that could be other than the percentage of organic carbon (5.3) over others. This substrate, however, bears promise for sustained use as it is easily available, and its development would be economically feasible in India as the sugarcane industry has, by now, attained an honourable status. The other reports on sugar refinery waste-based bacterial hydrogen produc- tion are those of Vincenzini et al. [15] and Vatsala [16].

In a previous report, we described potato starch-based H 2 production in BHU strain 1 [9], and ascertained that this bacterial strain also has amylase(s) to bring about starch breakdown to the level of simple sugars as re- ported in other nonsulphur photosynthetic bacteria [5, 6]. The observed enhancement of H 2 production in all the bacterial strains by potato starch coupled with the positive amylase test, certainly speaks of starch consump- tion by such microbes through amylase activity. The tests for amylase activity in the four bacterial strains were performed at normal (33°C) and elevated temperature (45°C) (Table 2). As expected, BHU strains 1 and 4 with high rates o fH 2 production, also exhibited a high degree of amylase activity compared with strains 2 and 3. The enzyme activity was suppressed in cells exposed to ele- vated temperature (45°C), with the exception of the thermotolerant strain (BHU strain 4).

The lowest enhancement in strains supplemented with untreated whey goes hand in hand with the percentage of total organic carbon (3.0), apart from the inability of R. capsulatus to convert lactose to hydrogen and its prefer- ence for lactic acid [8]. Such assumptions are based on the higher substrate conversion efficiency of Rhodospiril- lure rubrum over R. capsulatus when grown on undiluted whey. Cheese whey, a by-product of the dairy industry, contains milk-sugar lactose ( --. 5% w/v) and proteins in the range 0.8-0.9% as the major ingredients. We do not have the current estimates of whey produced by dairies in India, but certainly presume that a huge bulk is even- tually discharged in sewage as a waste. Salih [7] reported on whey-based H 2 production in R. rubrum S-l, and the

process could be enhanced if the whey was pretreated with lactose fermenting Escherichia coli. The latter inves- tigator observed a positive correlation between the lactic acid formed and the extent of H 2 output. It has been suggested that whey may also contain ingredients like citric acid, acetic acid, succinic acid and ethanol apart from lactose [17].

As the very habitat of all the bacterial strains prior to isolation suggested their frequent exposure to elevated temperature like 45°C during summer, it was of interest to also look into the hydrogen production efficiency at 45°C at the expense of all the three substrates compared before. In a previous report, we observed H 2 production by BHU strain 4 at elevated temperatures even without the addition of these substrates [10]. BHU strains 1, 2 and 3 in this regard, exhibited thermosensitive nature as evident from 58, 40 and 28% of H 2 production at 45°C, respectively, compared with those at 33°C. In contrast, the increased H 2 production in BHU strain 4 by 25%, ascertained its thermotolerant nature. In this case also, sugarcane juice supported the maximum H 2 production as all the strains showed an almost three- to four-fold increase over the control sets, followed by potato starch and whey in decreasing order. The overall data suggest that the agro-based product could also be used as substrate for achieving H 2 production by selected strains at elevated temperatures. The enhanced rate of H 2 pro- duction by BHU strain 4 over other strains at 45°C was statistically significant (F,t,,i, 3.9 = 17.54, P < 0.001) and also in terms of different substrates (F0~b.t,.te3.9 = 6.18, P < 0.001). There are also reports that a number of nonsulphur photosynthetic bacteria isolated from Thai- land exhibited almost equal rates of H 2 production at 30 ° or 40°C [18], Kim et al. [19] in a subsequent study, however, observed higher rates of H 2 production at 40°C only in R. sphaeroides B 5.

Immobilization of bacterial cells enhanced H 2 produc- tion and also stabilized the system for sustained meta- bolic activity [9, 15, 20, 21]. Since all the agro-based substrates used invariably favoured H 2 production in free cells of the bacterial strains used, experiments were extended to observe whether the same trend also contin- ued for immobilized cells. It is evident from the data presented in Table 1 that immobilization enhanced H 2

Table 2. A comparison of amylase activity in Rhodopseudomonas sp. (BHU strains 1-4)

Amylase activity*

Strain no. Source 33°C 45°C

BHU strain 1 Azolla root zone + + + + BHU strain 2 Salvinia root zone + + + BHU strain 3 Eichhornia root zone + + BHU strain 4 Deep-water rice plant + + + + + +

root zone

*Indicates the amylase activity in terms of maltose released.

440 S.P. SINGH et al.

production by nearly one-and-a-half times in all the four strains (F,,,~os.9 = 13.29, P < 0.005). Sugarcane juice, potato starch and whey supported H z production by various strains in the same decreasing order as noticed in case of free cells (F, ut~tr,t e 3,9 = 34.69, P < 0.001). Almost the same trend continued in the case where the immobi- lized cells were also exposed to 45°C (F,tr,~, 3.9 = 19.06, P < 0.001). Maximum H2 evolution, to the extent of 56.6 /~1 H2 rag- 1 dry weight, was observed in alginate-immo- bilized BHU strain 4 at 45°C among the all treatments. The variation in H 2 production by immobilized cells using different substrates could be verified statistically (Flu~,,te s.9 = 5.77, P < 0.05). Attempts are being made to enhance bacterial hydrogen production at the expense of various agro-based products outdoors.

Acknowledoements--The above work was supported by a re. search grant from the Department of Non-conventional Energy Sources, Government of India, New Delhi (Grant No. 103/8/86- NT).

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